Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography.

Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a "seamless" tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.

Three-dimensional imaging of podocyte ultrastructure using FE-SEM and FIB-SEM tomography.

Podocytes are specialized epithelial cells used for glomerular filtration in the kidney. They can be divided into the cell body, primary process and foot process. Here, we describe two useful methods for the three-dimensional(3D) visualization of these subcellular compartments in rodent podocytes. The first method, field-emission scanning electron microscopy (FE-SEM) with conductive staining, is used to visualize the luminal surface of numerous podocytes simultaneously. The second method, focused-ion beam SEM (FIB-SEM) tomography, allows the user to obtain serial images from different depths of field, or Z-stacks, of the glomerulus. This allows for the 3D reconstruction of podocyte ultrastructure, which can be viewed from all angles, from a single image set. This is not possible with conventional FE-SEM. The different advantages and disadvantages of FE-SEM and FIB-SEM tomography compensate for the weaknesses of the other. The combination renders a powerful approach for the 3D analysis of podocyte ultrastructure. As a result, we were able to identify a new subcellular compartment of podocytes, "ridge-like prominences" (RLPs).

Effects of Dietary Fish Oil and Apple Polyphenol on the Concentration Serum Lipids and Excretion of Fecal Bile Acids in Rats.

We studied the effects of fish oil and apple polyphenol combined with a high cholesterol diet in rats, and assessed serum and liver lipids concentrations, serum oxidative stress and fecal bile acid excretion. Young male rats were fed a diet containing the control (Control), apple polyphenol (AP), fish oil (FO) or fish oil+apple polyphenol (FO+AP) for 4 wk. The control diet contained a lard component. Posterior abdominal wall fat and testicle peripheral fat weights decreased in the FO+AP group compared to the AP group. The concentration of total cholesterol in the serum and liver decreased in the FO group and the FO+AP group compared to the Control and the AP groups. The concentration of adiponectin and biological antioxidant potential in the serum increased in the FO group compared to the other groups. The diacron-reactive oxygen metabolites in serum decreased in the FO group and the FO+AP group compared to the Control and the AP groups. The bile acid excretion in feces increased in the AP group, the FO group and the FO+AP group compared to the Control group. These results suggested that the combination of fish oil and apple polyphenol in the diet improved serum and liver lipids, which should assist in the prevention and improvement of metabolic syndrome.

Organ Specificity and Functional Relevance of the Arterial Structure: A Comparative Study in the Kidney and the Skeletal Muscle with Electron Microscopy.

The differences in circulation among various organs are well known, but the structural differences have only been poorly investigated. In the present study the wall structure of arteries was observed with electron microscopy in rat kidney and skeletal muscle. The wall thickness was almost equal in both organs at about 40 µm in luminal diameter, while it was relatively thin in the larger arteries and thick in the smaller arteries in the kidney compared with that in the skeletal muscle. The smooth muscle cells were regularly arranged in parallel in circular or slightly spiral orientation in the kidney, whereas those in the skeletal muscle were irregularly arranged in heterogeneous orientations. Extracellular matrices were more abundant in the arterial media in the skeletal muscle than in the kidney. The inner elastic lamina was continuous in the kidney, and arranged in longitudinal bundles in the skeletal muscle. The adventitial collagen fibers were abundant and dense in the skeletal muscle, and were scattered in small bundles in the fluid-filled spaces in the kidney. While the arteries in the skeletal muscle are under severe mechanical stress during muscle contraction and dilate dramatically during exercise, those in the kidney regulate glomerular pressure almost constantly.

Are the precapillary sphincters and metarterioles universal components of the microcirculation? An historical review.

The microcirculation is a major topic in current physiology textbooks and is frequently explained with schematics including the precapillary sphincters and metarterioles. We re-evaluated the validity and applicability of the concepts precapillary sphincters and metarterioles by reviewing the historical context in which they were developed in physiology textbooks. The studies by Zweifach up until the 1950s revealed the unique features of the mesenteric microcirculation, illustrated with impressive schematics of the microcirculation with metarterioles and precapillary sphincters. Fulton, Guyton and other authors introduced or mimicked these schematics in their physiology textbooks as representative of the microcirculation in general. However, morphological and physiological studies have revealed that the microcirculation in the other organs and tissues contains no metarterioles or precapillary sphincters. The metarterioles and precapillary sphincters were not universal components of the microcirculation in general, but unique features of the mesenteric microcirculation.

Structural arrangement of collagen fibrils in the periarterial connective tissue of the kidney: their functional relevance as a structural stabilizer against arterial pressure.

Periarterial connective tissue with a moderate amount of collagen fibrils is known to be a specialized domain in the renal interstitium. This study aimed to clarify the microscopic architecture of the periarterial connective tissue as a mechanical supportive structure of the intrarenal arteries. Transmission and scanning electron microscopy revealed two populations of collagen fibrils in the periarterial connective tissue. The major one was composed of many bundles of collagen fibrils running in longitudinal directions, whereas the minor one was represented by a few circumferential bundles adjacent to the smooth muscles. The amount of collagen fibrils was obviously variable and correlated with the arterial caliber. The correlation between abundance of collagen fibrils and the arterial caliber was confirmed by morphometric analysis of the collagen fibril area per arterial perimeter on electron micrographs. The size of individual collagen fibrils was measured in periarterial connective tissue of arteries with various calibers. A positive correlation between the diameter of collagen fibrils and arterial caliber was confirmed, indicating the supportive function of collagen fibrils in the periarterial connective tissue. The accumulated morphological findings supported the hypothesis that the collagen fibrils in the periarterial connective tissue develop longitudinal tension with their tensile strength, whereas the smooth muscle cells in the media develop circumferential tension with active regulation of contracting force.

Structural organization of pulmonary veins in the rat lung, with special emphasis on the musculature consisting of cardiac and smooth muscles.

Recent physiological studies have indicated the significant role of pulmonary veins in the total resistance of pulmonary vasculature. The structure of pulmonary veins in the rat was reinvestigated to clarify the different venous segments and their ultrastructure with regard to the musculature including cardiac muscles and smooth muscles with light and electron microscopy. The cardiac muscles were located in the axial and the primary branches of the pulmonary veins within a certain distance limit from the hilum (CM segment) and not in the peripheral region (non-CM segment). The smooth muscles were found indifferent to the presence of cardiac muscles as a continuous layer in segments larger than 180 microm (continuous SM segment) or as a discontinuous layer of circular smooth muscle cells in segments between 50 and 180 microm (partial SM segment). The smooth muscle layer was extremely thin in the CM segments, whereas it became conspicuously thick in the non-CM segment with an irregularly undulating luminal outline, especially in the partial SM segments. There were two elastic laminae in the CM segments: a conspicuous one on the interstitial side of the smooth muscles, and a weaker one between the endothelium and smooth muscles. In the non-CM segment, one elastic lamina was found on the interstitial side of the smooth muscles. Considering the limited range of contraction of cardiac muscles and the thinness of smooth muscle cells in the CM segments, it was concluded that vasoconstriction in the pulmonary veins is executed by smooth muscle cells in the non-CM segments thicker than 50 microm.

Mechanical components of rat intestinal villi as revealed by ultrastructural analysis with special reference to the axial smooth muscle cells in the villi.

The ultrastructure of the rat intestinal interstitium with regard to the mechanical components was analyzed from a functional viewpoint utilizing serial horizontal as well as longitudinal sections through the lamina propria mucosae, including both villi and crypts. The axial smooth muscle cells in the villi (villus-axial SMs) exhibited different configurations at various levels of the wall. They were separated from the voluminous fluid-filled spaces by sheet-like processes of fibroblasts in the upper part of the intravillous interstitium, formed a sheet around the central lymphatics, and were covered by the sheet-like processes of fibroblasts in the lower part of the intravillous interstitium. These villus-axial SMs were poorly developed and associated with the lymphatic walls in the upper part of the pericryptal interstitium; they were tapered and connected to microtendons composed of fascicles of longitudinal collagen fibrils in the lower part of pericryptal interstitium. At the apical termination, the villus-axial SMs were connected to myofibloblasts, which sent off many processes into the subepithelial meshwork layer of fine cell processes and extracellular matrices. The villus-axial SMs possibly develop longitudinal tension against the intravillous hydraulic pressure developing from the transepithelial absorption through the intestinal epithelium.

Structural and mechanical architecture of the intestinal villi and crypts in the rat intestine: integrative reevaluation from ultrastructural analysis.

The ultrastructure of the rat intestinal interstitium was analyzed from the viewpoint of mechanical dynamics to stabilize the intestinal villi, crypts and mucosal folds. In the rat, the small intestine lacks circular folds, but the large intestine possesses spiral folds. The intestinal villi, the largest in the duodenum, decreased in size in the jejunum and ileum successively, and were absent in the large intestine. The intestinal interstitium consisted of lamina propria mucosae (LPM) and tela submucosa (TSM) separated by muscularis mucosae (MM), the LPM was subdivided into an upper part within the villi and a lower part among the crypts in the small intestine. The light microscopic density of interstitium in the intestinal wall was lowest in the upper LPM, moderately dense in the lower LPM and highest in the TSM, and that among the intestinal region was highest in the duodenum and decreased successively in the jejunum and ileum. In the large intestine, the TSM bulged to form spiral folds with very low density. The intestinal epithelium in the villi possessed wide intercellular spaces and that in the crypts had closed intercellular spaces. At electron microscopic level, the upper and lower LPM contained subepithelial supportive meshwork that consisted of collagen fibrils and myofibroblast processes. The lower LPM and TSM contained conspicuous bundles of collagen fibrils and, in addition, TSM contained minor populations of scattered collagen fibrils near the smooth muscle layer (SML). The diameter of collagen fibrils was the largest in the bundles of TSM, and decreased from the duodenum through the jejunum and ileum to the large intestine. On the basis of these observations, we hypothesize that the intestinal villi are mechanically stabilized by the balance between the expansive interstitial pressure and inward pull by the subepithelial supportive meshwork. This hypothesis explains the hitherto neglected fact that the intestinal epithelium possesses wide intercellular spaces only in the villi, and accounts for the counterforce against the perpendicular smooth muscle cells, which are supposed to contract the intestinal villi.

The ultrastructure of periductal connective tissue and distinctive populations of collagen fibrils associated with ductal epithelia of exocrine glands.

The ultrastructure of the connective tissue around the intraglandular ducts was observed in rat exocrine glands. Connective tissue with a dense population of collagen fibrils was found either around the ducts and blood vessels (perivasculoductal connective tissue; PVDCT) as in the lacrimal and salivary glands and liver, or solely surrounding the ducts (periductal connective tissue; PDCT) as in the exocrine pancreas, whereas the interlobular and intralobular interstitium of the glands-except for the liver-contained substantially fluid-filled spaces without collagen fibrils. The PVDCT and PDCT of these glands contained two populations of collagen fibrils-fibroblast-associated and epithelium-associated-although the development and density of these fibrils varied considerably in individual glands. Both populations of collagen fibrils were most developed in the lacrimal glands, in which the basal aspects of the ductal epithelium and the basement membrane showed considerable undulation associated with a distinctive peribasement membrane zone with amorphous matter and a small population of the fibrils. In the parotid and submandibular glands, both populations were distinct, though poorly developed. In the exocrine pancreas and hepatic Glisson's sheath, the two populations of collagen fibrils were moderately developed, and the basal aspects of the ductal epithelium were characterized by prominent invaginations in which the multilaminar basement membranes and the epithelium-associated collagen fibrils were frequently engulfed. These observations provide evidence that the two populations of collagen fibrils around the ducts are found universally in exocrine glands, and support the hypothesis of the collagen fibril-synthesizing and -secreting ability of ductal epithelial cells.

Fibroblasts and antigen-presenting cells in the renal interstitium of streptozotocin-induced diabetic rats on a high cholesterol diet.

The cortical peritubular interstitium of the normal kidney contains both fibroblasts and antigen-presenting dendritic cells. Characteristics of these interstitial cells were analyzed in an overnutrition model by electron microscopy after the cold-dehydration technique and immunohistochemistry for antigen-presenting cells. In control rats, fibroblasts and dendritic cells were clearly identified by electron microscopy on the basis of their distinct ultrastructures. Fibroblasts possessed slender cell processes, and contained an abundance of actin filament bundles occasionally anchoring to surrounding structures, whereas dendritic cells possessed irregularly-shaped cell processes with a clear cytoplasm and a paucity of actin filament bundles. In the experimental kidney from diabetic rats given a high cholesterol diet, the peritubular interstitium contained fibroblasts and vacuolated cells, and the extracellular matrices such as collagen bundles were distinctly increased compared with the control rat kidney. Immunohistochemical staining with OX6 and ED1 revealed that the peritubular interstitium in the control rat kidney contained dendritic cells, while that in the experimental rats was occupied by macrophages. The present study provides the first evidence indicating that overnutrition may dramatically affect the immune cells in nonlymphoid tissue.